The Cropwatch Series

TAGETES

 

 

Tagetes Oil Phototoxicity – Further Clarifications 22/2/06

 

 

Copyright Ó Cropwatch Feb 2006

 

 

 

Proposed Tagetes Oil Restrictions: The Precautionary Principle

Gone Mad?

Amusingly, in an article in one of the leading aroma trade journals, Fenn (2005) recaps his fifty years trade experience which includes a couple of  industry’s top postings, and reproduces a quote that maintains that the EU Commission uses the precautionary principle - which “has been rejected by virtually all other governments - inappropriately.” The article further suggests that “the EU’s precautionary principle can over-rule the science-based decisions of regulators.” Let’s see how much potential science-based decision-making we can find for this particular issue and how the precautionary principle figures in the process…

Having examined the scientific literature more thoroughly Cropwatch concludes that there are no incidences of human phototoxicity which can be directly traced to the topical application of perfumes containing Tagetes oils. So you could even say that a cure (i.e. IFRA’s restriction of concentration of Tagetes oils to 0.01% in perfume compounds in its’ Voluntary Code of Practice 17th April 2001) has been invented for a problem that does not exist. In fact, Schmidt (1986) was brave enough to venture, perhaps with gardeners and bare-limbed countryside travellers in mind, saying that “…no authentic case of phototoxicity from plants known to contain a-terthienyl has yet been reported...”However, with the opportunity for more comprehensive examination of the scientific evidence via increasing sophistication of data-base scanning, there do appear to be a few recorded cases of sensitisation to Tagetes spp. from India & Africa.

 

Hausen & Helmke (1995) noted three phototoxic & sensitising compounds in an ether extract of marigold (from an unidentified Tagetes sp.) including the strong sensitiser a-terthienyl. Separately Meckes-Lozoya & Gaspar (1993) had earlier confirmed the phototoxic effects of methanolic extracts of Tagetes erecta. Interestingly, Jovanoviv & Poljacki (2003) discuss the belief that whereas thiophenes and acetylenes from Compositae elicit phytophotodermatitis (: a term used, for example, by Pathak 1986), in fact certain thiophenes (& benzofurans) possess separate phototoxic and sensitising properties. The authors further point out that photosensitivity is present in 22-75% of Compositae sensitive individuals. Cross-sensitivity with Tagetes in Arnica contact eczema has also been reported (Pirker C. et al. 1992). Because we feel more may yet be revealed in years to come about a possible connection between sensitisation and phototoxicity we will explore plant-induced sensitisation reports of Tagetes spp. in more detail. For example Warshaw & Zug (1996) discuss UV-light sensitivity and sesquiterpene lactone (SQL) allergy & Jojovanic & Poljacki (2003) suggest a mechanism for photoadducts between thymine bases on DNA and the a-methylene-gamma-lactone group of SQL’s. How this might tie in with components of tagetes oil such as a-terthienyl, which are both phototoxic and sensitising, or the fact that subjects who display UV-light SQL sensitivity also display tagetes oil sensitivity, have yet to be revealed.

 

Indian Experiences of Tagetes-provoked Contact Dermatitis.

Dr. Behl, Professor and Head of the Dept. of Dermatology at the M.A. Medical College & Irwin hospital New Delhi argues (Behl 1966) that “the leaves of the Tagetes spp. are known to irritate the skin of sensitive persons” (but gives no references). Singh (1978), also working at the same hospital department between 1976 and 1977 reported on all clinically suspected cases of plant-initiated contact dermatitis presenting at the hospital, which were subsequently screened and investigated (patch-tested). Out of 22,524 patients attending the dermatology clinic during this period, 618 (2.7%) dermatological problems were diagnosed as cases of contact dermatitis due to various contactants. Out of these, plants were deemed responsible for 90 cases and from these, just 3 cases tested positive to Tagetes patula.  All three were also tested for Parthenium hysterophorus (another plant of the Compositaea) but only one case showed simultaneous sensitivity (Singh 1978). Although the BoDD data-base does reference this data in abbreviated form (BoDD 2006), it is only by looking at the original source in detail that the extremely small scale of the occurrence of Tagetes contact dermatitis in this instance is apparent.

 

The Case of the Aromatherapist.

The only published paper citing a case of allergic contact dermatitis from Tagetes patula extract (wrongly cited in the title of the published paper as an essential oil) is in an aromatherapist (Paulsen 2002) as originally described by Bilsland & Strong (1990), who related a short case-history of a therapist with acute bilateral hand (palm) eczema who tested positive to 1.5% French marigold essential ‘oil’ in a grapeseed oil carrier. On closer examination of the scant information presented in the paper, we find that the tagetes “oil” was actually a commercially available acetone extract of the flower & leaves of T. patula, and not an essential oil at all, a fact apparently not picked up by the referees of the paper (which was published in Contact Dermatitis). Further, the subject had been spraying her roses with an insecticide product containing the synthetic pyrethroid permethrin 24 hours previously. The authors note that since “the initial acute episode followed the use of insecticide containing permethrin, a synthetic pyrethroid, this suggests cross-reaction similar to that noted with Tagetes minuta and pyrethrum (Chrysantheum coccineum),” referencing Verhagen (1974) – see below. You will note that the authors did not establish that the primary cause of the sensitisation was the Tagetes patula extract, and went on to suggest that permethrin, a synthetic pyrethroid (which the EPA has recently warned is carcinogenic & neurotoxic) produced an initial acute reaction, followed by patch-testing revealing to the authors, apparently, the existence of a cross-reaction connection with the T. patula extract (?). The authors cite this happened in a similar way to a Tagetes minuta extract that allegedly cross-reacted with natural pyrethrum from Chrysanthemum coccineum in the two examples provided by Verhagen (see below – but you will note that Verhagen found that the subjects that reacted positively to T. minuta extracts, did not in fact react to extracts from T. patula). Bilsland & Strong’s conclusions therefore seem to lend more to speculation than to robust science!

 

We further examine another of Bilsland & Strong’s statements, which leads us to consider material in a leading text on botanical dermatology in more detail:
 
1. Bilsland & Strong give a reference to back up the claims that "There are a few reports of contact dermatitis attributable to Tagetes species” and then say contradictorily in the next sentence: “Tagetes minuta is a common cause of dermatitis in South Africa” (Mitchell &, Rook 1979). Cropwatch could not find any reference to Tagetes/S. Africa in Mitchell & Rook’s large volume on toxic plants - in fact there are just three mentions of Tagetes referenced in the book, and only one mention of the word Tagetes specifically found in the text. Further, the statement that T. minuta is a common cause of dermatitis in S. Africa cannot be substantiated by extremely low numbers of cases reported in the literature. 

 

2. On p176 Mitchell & Rook classifiy marigolds (Tagetes) into the ‘tribe' of Helenieae and separately claim that “the odour of marigolds caused hayfever and asthmas in two patients respectively” and that “Crown of Gold Marigold” which had odourless foliage had no ill-effect (Biederman 1973)” perhaps insinuating that the absence of recognisable odour volatiles corresponds to the absence of an adverse reaction.

 

3. On p174 under the heading of Compositae, Mitchell & Rook describe the Heliantheae tribe as providing the largest number of plants reported to cause dermatitis and announces that “The major sensitising agents of the family are sesquiterpene lactones and the distribution of these chemicals in the plants result in cross-sensitivity patterns between species. Consequently, a patient contact-sensitive to sesquiterpene lactones will frequently show positive patch test reactions to plants to which a history of exposure is lacking.” Unfortunately no references are given for this assertion [Cropwatch should point out that sesquiterpene lactones are not generally found in Tagetes oils].

 

The passage above is essentially supposed to be a discussion of the botanical Family to which Tagetes belongs (the Compositae) not the Tagetes Genus specifically, or any particular Tagetes spp.  It is a mystery therefore why Bilsland and many of her contempories make assertions regarding accounts of contact dermatitis for Tagetes minuta, and in the same breath talk about the causation for chemical sensitivity or adverse reactions in the  Compositae family as being sesquiterpene lactones. It is this muddled presentation which gives casual readers the impression that all Tagetes spp. show sensitisation because of contained sesquiterpene lactones (which you will remember are largely absent in Tagetes spp.). This is discussed further in the Aromatherapy section below.
 

The Kenyan Experience with Tagetes-provoked Sensitisation.

Verhagen reports on a female farmer presenting with “dermatitis of the exposed parts suggesting that her condition might be caused by a weed, which was later determined to be Tagetes minuta L. or Mexican marigold. She absconded after a strongly positive patch test.” After this incident, T. minuta was routinely included in patch tests on patients with dermatitis of the exposed parts (Verhagen 1974).  Verhagen further reports on two cases of positive testing for plant contact allergy to T. minuta in two male farmers, again in the Kenyan highlands. The author notes that “the skin reactions depended on the season and vigour of the specimens, as when dried or preserved even for a few days, the effects of Tagetes minuta on the skin disappear”.

 

Initial patch tests with intact leaf and flowerhead gave a positive result within 24 hours. The author then extracted Tagetes minuta flowerhead & leaf samples with dilute mineral water, dilute alkali, diethyl-(?)ether and acetone, and neutralised and concentrated the material. As with the fresh plant, the ether and acetone extracts produced a maximal patch test reaction with severe edema, erythema and vesiculation, extending far outside the patch and having a diameter of 5 to 8 cm. Tests with the other extracts and extracting solvents were negative. The 3% w/v (?) solution of marigold plant material in acetone, which gave the strongest reaction, was further diluted stepwise by adding the same volume of the solvent to the previous dilution. The highest dilution that produced clearly positive reactions in the two patients was 1/64 and 1/256 respectively for the extract from the flowerhead and 1/32 and 1/128 respectively for the leaf extract. Out of 32 controls tested with flower and leaf extract, 22 produced a positive reaction (though not always in the same patient) The 3% acetone extracts, applied on 11 patients, produced eight positive reactions to the flower extract and six to the leaf extract. Further dilutions on six controls showed that the highest further dilution that produced a positive reaction was ¼ for the flower head in one case and ½ for the leaf in two cases. All reactions in controls were morphologically different to those of the patients tested and invariably consisted of a minute, sharply marginated blister or of mild erythema, strictly limited to a part of the area of the patch and were positive, the other solvents negative with the strongest being the 3% solution of the Tagetes in acetone. (Verhagen 1974).

 

Acetone flower and leaf extracts of two “known sensitisers” from the Compositaea family, Chrysantheum morifolium and pyrethrum were also tested and both patients reacted mildly to the C. morifolium flower extract, but not the leaf, and one of the men reacted strongly to the pyrethrum and the other not at all. Both extracts gave no positive reactions in controls and interestingly, patch tests with Tagetes erecta cultivars also were negative.

 

In contrast to Bilsland  & Strong’s submission that the use of insecticide containing the synthetic pyrethroid permethrin that preceeded the acute episode experienced by the aromatherapist  suggested cross-reaction similar to that noted with Tagetes minuta and pyrethrum (Chrysantheum coccineum),”  Verhagan actually notes in his  paper that in the case of the two farmers, cross-reaction “could practically be excluded”, as neither of the Compositae spp. above, nor pyrethrum, is cultivated in the area where the reaction took place.  Verhagan did exclaim the following though “it is probable that in other geographical areas such cross-sensitivity to better known sensitizers might have been detected and the cases might have been diagnosed as chrysanthemum or pyrethrum dermatitis.”

 

The example of the two farmers is one of the few documented cases where Tagetes minuta has been shown ‘beyond reasonable doubt’ to be the main sensitizer. The two cases previously described involved men working in close contact with vegetation (20 large plants/m2) for 5 and 13 years respectively at an altitude of 6,000 ft, in an area where Tagetes minuta is not a native plant and reactions were experienced during the “rainy season”. However, specifically concerning phototoxicity - in these two particular cases UV-A tests were also performed, irradiating patch test sites with a radiation dose that represented “three to four times the average minimal erythema dose in Africans” - in these tests, no difference was seen between irradiated and covered patch tests in patients and controls.

 

The conclusions are unclear. It remains apparent that statistically a very few watertight cases of pant-induced contact sensitivity from Tagetes spp. (where Tagetes is clearly identified as the causative agent) have been found. It also appears on the evidence above that acetone & ether extracts of fresh Tagetes minuta flowers or leaves will provoke sensitisation reactions  (if concentrated enough) in a significant number of individuals – although we are unclear from this account about the identities of the causative agents. 

 

On a different matter, the phototoxic effects of a-terthienyl which occurs at low level in oils from Tagetes spp. (see below under a-terthienyl for further information) is reported by several authors (human skin: Chan et al. 1977; Towers et al. 1979; and for guinea pig skin: Rampone et al. 1986). This is explored further under the a-terthienyl section below.

 

Aroma therapeutic Uses of Tagetes Oil.

Tagetes oil is not a particularly popular essential oil for general use in aromatherapy - the main reasons being its “phototoxic” reputation, the ever-growing popularity/proven efficacy/availability of Tea tree oil as a safer alternative for anti-fungal purposes, and the fact that the characteristic aroma of the oil can be quite overwhelming or bordering on the unpleasant, except in extreme dilution.

 

So, when tagetes oil is used in aromatherapy, it is often for its anti-fungal action – for example Kyle (1998) indicates its use as a treatment for nail-fungus. A non-scientific survey by Cropwatch of a small sample of clients receiving this treatment from aromatherapists has revealed no subsequent reported adverse effects to date, but a larger and better-designed survey is needed to establish this conclusively (this unfortunately is still on Cropwatch’s “to do” list at the present time!). Finding enough subjects who have had an intervention with tagetes oil to make the data meaningful, is expected to be a problem.

 

Other topical uses of tagetes oil include the treatment of warts & veruccas where it may be used neat directly onto the affected area but more often it is used diluted (between 20% and 50% concentration in a vegetable carrier oil vehicle is common). Cropwatch believes it has identified a present trend amongst aromatherapists towards combining tea tree oil with tagetes oil (in carrier approximately at the dilutions mentioned) to give wider anti-fungal effect. It should be made clear that when aromatherapists talk about  ‘Marigold oil’ they will often mean the more commonly used Calendula oil/ extract from Calendula officianalis L. (see below) and the botanical origin of the oil needs to be clearly ascertained in any meaningful investigation.

 

It is common to find in Aromatherapy courses and text books, references to the “toxic or neurotoxic nature of Tagetes oil, usually (incorrectly) citing the sesquiterpene lactones present (we repeat, these are in fact absent in Tagetes oils) and/or also the ketone tagetone as the main culprits (see IJA 1977 for comment on tagetone’s alleged toxicity). In the case of the two Kenyan farmers quoted above by Verhagen, no positive reactions were shown with the application of the sesqiterpene lactone alantolactone at 1% concentration. As Verhagen states (alantalactone) is “one of the numerous sesquiterpene lactones incriminated as sensitisers in Compositae”, asking for further study. It might be true that there is a tenuous connection between sesquiterpene phototoxicity and a-terthienyl phototoxicity (such a hypothesis has been advanced as mentioned above) but here we are getting into virtually unexplored waters…

 

Muddled Identification of Tagetes Commodities. 

General confusion over labelling of medicinal herbs has been previously reported (Linares & Bye 1987). Specific confusion over the term ‘Marigold’ is very common – qualities from Calendula officinalis L. (Common, Scotch or Pot Marigold) commonly being confused with Tagetes oil species such as Tagetes minuta L. (African Marigold) in perfumery & herbalism as well as in aromatherapy as noted above.

 

As Cropwatch reported previously, significant differences in essential oil composition occur throughout the oil bearing Tagetes species (Lawrence 1985; Zygaldo et al. 1993), with Craven et al. (1991) reporting that soil type & nutrient status affects the composition. But Cropwatch maintains when perfumers and essential oil traders refer to tagetes oil, they mean an oil high in content of acyclic monoketonic ketones represented by tagetones & ocimenones, probably derived from the aerial parts of the plant. They do not necessarily mean Tagetes flower oils containing elevated levels of piperitone & piperitenone, or yet other Tagetes oils which might contain major components such as methyl chavicol – found in the oil of T. filifolia ssp. filifolia (Bohrmann & Youngken 1968). In partial support of this hypothesis, Ohloff (1994) ventures that the characteristic odour of tagetes oil is ascribed to tagetone, dihydrotagetone & (Z)-ocimenone, which are present at over 50% in tagetes oil.

 CONTINUED IN RIGHT-HAND COLUMN

 

References

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Amason J.T., Chan C.F.Q., Wat C.K., Downum K.R. & Towers G.H.N. (1981) Photochem. Photobiol. 33, 821-824.

 

Bakker J., Grommers F.J. Nieuwenhuis I. & Wynberg H. (1979) J. Biol Chem. 234, 1841-1844.

 

Behl, P.N., Captain, R.M., Bedi, B.M.S. & Gupta, S. (1966). Skin-Irritant and Sensitizing Plants found in India, New Delhi. Pub. P.N. Behl, Irwin Hospital. Asian Printers, Bombay, India. 

 

Bernhard-Smith, A. (1923). Poisonous plants of all countries. 2nd ed. London, England.

 

Bilsland D. & Strong A. (1990) “Allergic contact dermatitis from the essential oil of French Marigold (Tagetes patula) in an aromatherapist.” Contact Dermatitis 23(1), 55-6.

 

BoDD (2006) – see http://bodd.cf.ac.uk/BotDermFolder/BotDermC/COMP-20.html

 

 

Bormann H. & Youngken H.W. (1968) “Estragole, the main compound in the volatile oil of Tagetes filifolia (Compositae)” Phytochem 7, 1415-1416.

 

Chan G.F., Prihoda M., Towers G.H. & Mitchell J.C. (1977) “Phototoxicity evoked by alpha-terthienyl” Contact Dermatitis 3(4), 215-6.

 

Chan., G.F.Q, Towers, G.H.N., Mitchell, J.C. (1975). “Ultraviolet-mediated antibiotic activity of thiophene compounds of Tagetes.” Phytochemistry 14, 2295-2296.

 

Chisowa E.H., Hall D.R. & Farman D.I. “Chemical composition of the essential oil of T. minuta from Zambia” J. Essen. Res. 10, 183-184.

 

Craven E.H., Webber I., Benians G., Venter M. & Gardener G. (1991) “Effect of Soil Type & Nutrient Status on the yield & composition of Tagetes oil (T. minuta) J. Essen. Oil Res. 3, 303-307.

 

De Feo V., Soria E.U., Soria R.U & Pizza C. “Composition & in vitro toxicity of the essential oil of Tagetes terniflora HBK (Asteraceae).” Flav. & Frag. J. 20, 89-92.

 

Downum K.R. & Towers G.H.N “Analysis of thiophenes in the Tagetaceae

(Asteraceae) by HPLC.” J. Nat Products 48, 98-103.

 

Fenn, R.S. (2005) “The Four P’s: Perfumery – past, present & prologue” Perf. & Flav. 30, Jan/Feb 2005 p18-24.

 

Héthelyi É., Dános B. & Tétényi P. & Koczka I. (1986) “GC/ MS Analysis of essential oils of some Tagetes species.” Progress in Essential Oil Research Walter de Gryper, Berlin.

 

Joshi V.P., Singh B., Kaul V.K. & Mahmood U. (2005) “Chemical transformation of 3,7-dimethyl,5-one,1-octene (dihydrotagetone) into new odour molecules.” Flav & Frag, J. 20, 592-595.

 

Hausen R.M. & Helmke H. (1995) “Butenylthiophene, alpha-terthienyl & hydroxytremetone as contact allergens in cultivars of marigold (Tagetes sp.)” Contact Dermatitis 33(1), 33-7.

 

IJA (1977) – “Letters” International Journal of Aromatherapy 8(1), 35-37.

 

Jacobs J.J., Aaroo R.R., De Koning E.A., Klunder A.J., Croes A.F. & Wullems G.J. (1995) “Isolation & characterisation of mutants of thiophene synthesis in Tagetes erecta.Plant Physiol. 107(3), 807-814.

 

Jovanovic M. & Poljacki M. (2003) “[Compositae Dermatitis”]. Med. Pregl. 56(1-2), 43-49.

 

Kagan J., Kagan E.D & Seigneurie E (1986) “Alpha-terthienyl, a powerful fish poison with light-dependent activity” Chemosphere 15(1), 49-57.

 

Krishna A., Kumar S., Mallavarapu G.R. & Ramesh S. (2004) “Composition of the essential oils of the leaves & flowers of Tagetes erecta L.” J. Essen. Res. 16, 520-522.

 

Kyle L. (1998) “Aromatherapy for Elder Care” International Journal of Aromatherapy 9(4) 170-177.

 

Lawrence B.M. (1985) “Essential Oils of the Tagetes Genus” Perf. & Flav. Oct/Nov 1985 pp73-85.

 

Linares E, Bye RA Jr. (1987) “A study of four medicinal plant complexes of Mexico and adjacent United States.” J Ethnopharmacol. 19(2), 153-83.

 

Maiden JH (1895) “The weeds of New South Wales. Supplementary notes. No. 1”. Agricultural Gazette of New South Wales 6(10), 671-678.

 

Marles R., Durst T., Kobaisy M., Soucy-Breau C., Abou-Zaid M., Arnason J.T., Kacew S., Kanjanpothi D., Rujjanawante C., Meckes M. et al. (1995) “Pharmokinetics, metabolism & toxicity of the plant-derived phototoxin alpha-terthienyl” Pharmacol Toxicol. 77(3), 164-8. 

 

McLachlan D, Arnason J.T. & Lam J. (1984) Photochem Photobiol 39, 177-182.

 

Merckes-Loyoza M. & Gaspar I. (1993) “Phototoxic effects of methanolic extracts from Podophyllum macrocephalum & Tagetes erecta.” Fitoterapia 64(1),35-41.

 

Mitchell J. & Rook A. (1979) Botanical dermatology: Plants injurious to the skin. Vancouver: Greengrass

 

Mohamed M.A., Harris P.J., Henderson J. & Senatore F. (2002) “Effect of drought stress on the yield & composition of volatile oils of drought-tolerant & non-drought-tolerant clones of Tagetes minuta.” Planta Med. 68(5), 472-4.

 

Nivsarkar M., Kumar G.P., Laloraya M. & Laloraya M.M. (1992) Arch. Insect Biochem. Physiol. 19, 261-270.

 

Nivsarkar M., Kumar G.P., Laloraya M. (1996) “Metal binding & resultant loss of phototoxicity of a-terthienyl: metal detoxification vs. a-terthienyl inactivation.” Bull. Environmental Contam. Toxicol. 56, 183-189.

 

Nivsarkar M. (1999) “Identification of a-terthienyl radical in vitro: A new aspect of alpha-terthienyl photoxicity” – see http://ns1.ias.ac.in/currsci/may25/articles32.htm

 

Nivsarkar M. (2001) “Alpha-terthienyl: A plant-derived new generation insecticide” Current Sci. 81(6), 667-672.

 

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Weaver D.K., Wells C.D., Dankel F.V., Bertsch W., Singh S.E. & Sirharan S. (1994) “Insecticidal activity of floral, foliar and root extracts of Tagetes minuta (Asterales: Asteraceae) against adult Mexican bean weevils (Coleoptea: Bruchidae).” J. Econ. Entomol. 87, 1718-1725.  

 

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Zygaldo J.A., Abburra R.E., Maestri D.M., Guzman C.A., Grosso N.R,, Azira Espinar L. Flav & Frag. J. 8, 273-275.

 

Zygaldo J.A. et al. (1990) “Essential oil variation in Tagetes minuta populations.” Biochem Systemat. Ecol. 18, 405-407 (1990).

 

 

 

 

 

 

 

 

 

It is particularly worth mentioning that ISO 4720 (Essential oils – Nomenclature) 2nd edn (2002) defines Tagetes oil as coming from Tagetes minuta L. (only).  This seems pretty sensible to Cropwatch; lumping in oils from other Tagetes spp. as tagete oil without separately defining them by common name and geographic origin, or by other means, can only confuse the issue. Four Tagetes oils from species common as garden ornamentals throughout the world (Lawrence 1985) are listed below (as well as two additional Tagetes spp.) but Lawrence provides details of a total of some forty essential oils isolated from different Tagetes spp.

 

Other more minor use commodities from Tagetes spp. (concretes, absolutes, oleoresins etc.) present a more obscure picture with respect to available analytical information, and will not be considered further.   

 

Tagetes erecta L. (African Marigold; Mexican Marigold). CAS No: 90131-43-4.

This is a poor-yielding essential oil containing species, common as a garden ornamental & previously listed on the EU Inventory of Fragrance Ingredients. The presence of thiophenes in the oil of T. erecta has been shown by Zechmeister & Sease (1947), Uhelbroek & Bijloo (1958), and Downum & Towers (1983). Jacobs et al. (1995) describe T. erecta mutants with aberrant thiophene biosynthesis which contained high amounts of the C13 monothiophene 2-(but-3-en-I-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type.

 

Krishna et al. (2004) reviewed the literature on the composition of leaf & flower oils of T. erecta (citing 9 references) and analysed the oils from T. erecta plants grown near Lucknow. The Indian capitula oil contained piperitone (28.5%), piperitenone (10.9%) and Z-myroxide (7.9%) whereas the leaf oil was found to contain piperitone (52.4%), terpinolene (11.9%) and limonene (7.6%) as major constituents. Tagetenones were pretty well absent in the capitula oil and only present at 1.9% (total isomers) in the leaf oil. Conversely Singh et al. (2003) found the major components in the hydrodistilled leaf oil from T. erecta grown in Kushinigar, India analysed by GC-MS consisted of (Z)-b-ocimene (42.2%), dihydrotagetone (14.3%) & (Z)-tagetone (8.3%) with no listing of piperitenone or piperitone.

 

Tagetes lucida Cav. (Mexico, Guatemala) Sweet Scented Marigold.

Little reliable analytical data available. As previously mentioned, Héthelyi et al. (1986) found Hungarian steam-distilled oils contain 16.5% limonene, 13.8% b-ocimene, and 28.0% b-caryophyllene.

 

Tagetes minuta L. (S. America; South Africa) syn. T. glandulifera Schrank (this is disputed) syn. T. glandulosa Schrank. CAS No: 91770-75-1.

T. minuta has now become widespread throughout the world (Lawrence 1985), and is listed on the EU Inventory of Fragrance Ingredients.

 

Major constituents of the oil of T. minuta are (Z)-b-ocimene, dihydrotagetone, tagetones (E- & Z-) and ocimenones (E- & Z-) according to Joshi et al. (2005) who also suggest that the dihydrotagetone content [(15-30%) grown in temperate conditions and up to 48% at lower altitudes] varies with altitude. Chisowa et al. (1998) report that T. minuta essential oil grown in Kitwe, Zambia has of dihydrotagetone (30.0%), Z-b-ocimene (23.6%) and (Z)-tagetone as principal components. Mohamed et al. (2002) have also shown that drought stress affects the yield and composition of T. minuta oils.

 

Downham & Towers (1983) reported the presence of four thiophenes in the oil. Wells (1993) found these thiophenes in a methylene chloride extract of plants grown in Alabama:

 

5-(but-3-en-1-ynyl)-2,2’-bithiophene (0.15%)

5-(but-3-en-1-ynyl)-5-methyl-2,2’-bithiophene (0.03%)

2,2’,5’,2’’-terthiophene (0.36%)

5-methyl-2,2’,5’,2’’-terthiophene (0.20%)

5-(4-acetoxy-1-butynyl)-2,2’-bithiophene (0.03%).

 

As previously mentioned, Weaver (1994) reports on a T. minuta leaf oil which contains 47.5% dihydrotagetone, 9.6% limonene and 6.0% as (E)-tagetone but also 2.30% 5-methyl-2,2’,5’,2’’-terthiophene and 0.4% a-terthienyl. The flower oil contained 31.9% of (Z)-b-ocimene, 2.1% a-terthienyl & 1.0% 5-methyl-2,2’,5’,2’’-terthiophene

 

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Tagetes patula L. (French marigold). CAS No: 91722-29-1.

This is a poor-yielding essential oil containing species, listed on the EU Inventory of Fragrance Ingredients. a-Terthienyl was confirmed as a component of the oil by Uhelbroek & Bijloo (1958), the same authors confirming the generation of 5-(3-buten-1-ynyl)-2,2’-bithienyl from the plant roots a year later Uhelbroek & Bijloo (1959). Sutfield (1982) found small amounts of thiophene in the steam distilled essential oil of T. patula and Downam & Towers (1983) confirmed the presence of four thiophenes.

 

Margl et al. (2002) confirmed the presence of a number of thiophenic compounds (BBT, PBT, BBTOH, BBTOAc etc. – see key) by GC & GC-MS in diverse plant organs and in vitro root, callus & cell suspensions of T. patula L. cv. Carmen. The authors noted that only the root cultures produced substantial amounts of irregular thiophenes in vitro. Romagnoli et al. (2005) report the composition of a steam-distilled oil from the capitula of T. patula, which shows low levels of tagetones (dihydrotagetone 4.91%; cis-tagetone 4.62%) and higher levels of piperitone (24.74%) and piperitenone (22.93%)

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Tagetes terniflora HBK.

Feo et al. (2005) report on the composition of the aerial parts of the essential oil of this S. American herb oil used in folk medicine in Peru, reporting tagetones (22.4%), ocimenes (20.6%) and ocimenones are major components. Zygaldo et al. (1993) analysing essential oil from the aerial parts & the inflorescences of the same species found cis-tagetone contents >67%.

 

a-Terthienyl.

Nivsarkar (1999) summarised the properties of the naturally occurring larvicide a-terthienyl, which is found in abundance in the roots of many Tagetes species. The author remarks on its phototoxicity, pointing to its absobtion peak @351nm in ethanol, its ability to generate singlet oxygen (McLachlan et al. 1984), and the superoxide anion radical, both in vivo (Bakker et al. 1979; Nivsarkar et al. 1992) and in vitro under UV-A light (Amason et al. 1981; Nivsarkar et al. 1992). Nivsarkar (1999) further showed, via electronic paramagnetic resonance studies, that a-terthienyl could also generate the a-terthienyl free radical on exposure to UV light, which transformed to the a-terthienyl superoxide anion radical. The author goes on  to point out the damage done by free radicals to biological membrane systems and the likelihood of damage to non-target systems exposed to it. In a further article, Nivsarkar  (Nivsarkar 2001) describes a-terthienyl as a safe and rapidly biodegradable insecticide (with a half-life of 4 hours in sunlight according to Philogene 1985) compared with organochlorine and organophosphate insecticides that persist in the environment (and of course bioaccumulate. However dermal studies with 1% terthienyl in a petroleum vehicle show a different perspective. When exposed to UV light erythema, blistering and hyperpigmentation were apparent in a biphasic respose.Chan et al. (1975), Towers et al. (1977), Chan et al. (1977) Towers et al. (1979). In the investigations of the latter workers, associated polyacetylenes applied together with a-terthienyl did not appear to evoke a separate reaction. Rampone et al. (1986) demonstrated dose-dependent cutaneous phototoxicity of the epidermis, adexae and superficial dermis in guinea pigs with 0.1% and 1% a-therthienyl applied in a non-irritating 3% Azone gel vehicle, obtaining similar results using 5-500mg a-terthienyl injected intradermally.

 

In an earlier study (Nivsarkar 1996) it was demonstrated that a-terthienyl is able to complex with cobalt (Co2+) ions, and that the complex was found non-toxic to four different mosquito larval stages (results not presented) as opposed to 100% mortality with straight a-terthienyl.

 

In other respects a-terthienyl shows toxic effects, including functioning as a strong UV light-activated fish poison twice as potent as rotenone (Kagan et al. 1986). Marles et al. (1995) have determined the LD50 of synthetic a-terthienyl interperitoneally (rats) as 110mg/Kg, but was found to be non-toxic when fed orally to rats at 0.1%. Pharmokinetic and metabolic studies were also carried out.

 

Possible Regulation of Thiophene Biosynthesis in Tagetes spp.

Aaroo et al. (1997) discuss the biogenesis of thiophenes (BBT, BPT, BBTOAc, MeBBT etc. - see key) in Tagetes spp., revealing that reduced sulphur species add to adjacent acetylenic groups.  Ultimately the authors suggest that the S atom comes from sulphate in the soil, and they were able to show in Tagetes patula plants that a 20 to 40 fold reduction in sulphate availability did not affect several growth criteria but decreased thiophene levels 20-50%, becoming restored on transfer to the standard medium, but being repressed by corycepin, an inhibitor of mRNA processing. Clarkson et al. (1983) had previously indicated that sulphate is accumulated in the plant as a pool until the medium is exhausted, and is sufficient to maintain growth & development of Tagetes roots for a long period although it is insufficient for thiophene production.

 

It may be reasonable to speculate therefore that low thiophene containing Tagetes essential oils suitable for cosmetic use (because of their reduced phototoxic potential ?) could be produced by agrotechniques that control the sulphate level, such as hydroponics, provided the restricted sulphate level does not interfere with the yield of essential oil production. Cropwatch has put this proposition to two organisations concerned with the promotion of essential oil bearing plants.

 

Further, the possibility that commercial UV absorbers, common in many fragrance compounds where light stability (absence of discolouration on light exposure etc.) is required, could prevent the light-activated phototoxic action has not been explored, as far as Cropwatch is able to establish.

 

Conclusions. Cropwatch stands by its’ earlier conclusions, additionally concluding:

 

  1. The incidence of reported Tagetes phototoxicity caused by fragrances containing ingredients from this spp. are zero in the scientific literature.

  2. Existing IFRA (2001) guidelines on the limiting concentration of Tagetes oil to 0.01% in fragrance compounds is completely adequate. The statement in the SCCP Opinion SCCP/0869/05 that there is no safe limit of Tagetes oil in cosmetics is unsafe, and another example of excessive regulation resulting from inappropriate deployment of the precautionary principle.
  3. Formulations of Tagetes oil containing perfume compounds and dilutions of Tagetes oils with and without commercial UV absorbers should both be independently tested to see if the latter compounds eliminate any phototoxic effects on human skin.
  4. Tagetes oil producers may anyway be able to produce thiophene-reduced or thiophene-free essential oils, by limiting available sulphate levels. However, whilst it may be tempting for regulators to regard the photo-labile substance a-terthienyl as a surrogate substance for tagetes oil, there is, as yet, no evidence that this would be anything but an inappropriate course of action.

 

 

 

 

 

 

Glossary.

Perfume compound:    (as used in the trade) a completed perfume formulation

                                     i.e. the perfume concentrate before addition of alcohol.

(E)- :                             trans-  e.g. (E)-ocimenone = trans-ocimenone

(Z)- :                             cis-      e.g. (Z)-tagetone = cis-tagetone

 

 

 

Abbreviations Key

BBT = 5-(but-3-en-1ynyl)-2,2’-bithiophenyl-5’methylacetate

BBTOH = 5-(4-hydroxy-1-butynyl)2,2’-bithiethyl)

BBTOAc = 5-(4-acetoxy-1-butynyl)2,2’-bithiethyl)

BPT =  5-(but-3-en-1ynyl)-5-(penta-1,3-diynyl)-thiophene

MeBBT = 5-(but-3-en-1ynyl)-2,2’-bithiophenyl-5’-methane